Falciparum malaria parasites sequester in placentae and imperil millions of pregnant women and newborns each year. Sequestration is mediated by P. falciparum VAR2CSA proteins inserted on the erythrocyte surface which bind to placental chondroitin sulfate A (CSA), produce placental inflammation, and result in low birth weight (LBW) babies. Adaptive immunity to VAR2CSA protects pregnant women and newborns from malaria morbidity. VAR2CSA is large and diverse with incompletely described antigenic and immunogenic domains, but one 2.4 kb region of var2csa, encodes ID1-DBL2X-ID2, which binds CSA with high affinity when expressed in vitro. This recombinant protein elicits potent binding inhibitory antibodies and thus has great promise as a vaccine candidate. However, because VAR2CSA is highly diverse, a polyvalent vaccine might be necessary. Here we propose to interrogate var2csa ID1-DBL2X-ID2 diversity and identify potentially pathogenic variants in Malawian pregnant women, and then measure the CSA binding and immunogenicity of expressed recombinants. 200 pregnant women with both 2nd trimester and delivery parasitemias will be selected from a 1600-person trial comparing Intermittent Screening and Treatment (IST) with Intermittent Preventive Therapy (IPT). They will be divided into 4 groups: primigravidae with and without low-birth weight (LBW) offspring and multigravidae with or without LBW offspring. From each woman, we will obtain 3 parasite samples: peripheral blood from the 2nd trimester and from delivery, and from placental blood. For the first aim, each sample type from each group will be pooled (12 pools) and deep-sequenced across the ID1-DBL2X-ID2 region. We will identify variants associated with primigravidity and LBW. In the second aim, we will use qPCR and reverse-transcriptase qPCR to compare population sizes of the 50 most common variants within each woman over the course of pregnancy. We hypothesize that the most pathogenic variants will be more common in the 2nd trimester than at delivery. In the third aim, we will synthesize and express recombinants of the variants that best fit these criteria and measure both their in vitro CSA-binding and immunogenicity. This proposal represents a new collaboration between the UNC group, with expertise in epidemiology and deep sequencing, and the University of Copenhagen group, a leader in var2csa biology and vaccine development. This proposal will help elucidate the pathogenesis of malaria in pregnancy and directly inform the on-going development of a vaccine against pregnancy-associated malaria.